Uses of hypoxia-inducible factor inhibitors

ABSTRACT

The present invention relates to treating a hematologic cancer using a Hypoxia-Inducible Factor (HIF inhibitor). The invention also relates to inducing acute myeloid leukemia remission using the HIF inhibitor. The invention further relates to inhibiting a maintenance or survival function of a cancer stem cell (CSC) using the HIF inhibitor.

FIELD OF THE INVENTION

The present invention relates to treating hematologic cancers.

REFERENCE TO THE SEQUENCE LISTING

Applicant hereby makes reference to the Sequence Listing that iscontained in the file “SL.txt (6 kB; created on Aug. 6, 2010), thecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

It is estimated that 12,800 new cases acute myeloid leukemia (AML) willbe reported in 2009, with 9000 deaths. Although complete remission canbe achieved in most cases through chemotherapy, prolonged remission orcure is rare. Accordingly there is a need in the art to treat AMLpostremission. Postremission leukemia, however, tends to be moreresistant to chemotherapy in general. Underlying reasons for thisinclude expression of the multi-drug resistance protein Pgp1, andpossible residence in a bone marrow area that is beyond the reach ofdrugs.

AML has been hypothesized to be associated with cancer stem cells (CSC).This idea is supported by phenotypically identifiable CSC subsets in AMLcells, and the efficacy in testing CSC in an AML model of both in vitrocolony-forming units (CFU) and xenogeneic transplantation models.

Many human cancers besides AML contain CSC that are considered to beresponsible for driving and maintaining tumor growth and resistance totherapy. Understanding the mechanism of self-renewal of CSC is thereforenot only crucial for understanding the fundamental mechanism of cancerdevelopment, but also provides new approaches for long-lasting cancertherapy. Much like normal stem cells, self-renewal of CSC involves tworelated processes. First, the stem cells must undergo proliferation toproduce undifferentiated cells. The known pathways for self-renewal ofnormal and cancer stem cells, including Wnt and Hedgehog, regulate theproliferation, at least in part by controlling the expression of Bmi-1,a critical regulator for proliferation of normal and cancer stem cellproliferation. Second, the CSC must survive in an undifferentiated statethroughout tumorigenesis. Survival of CSC may underlie difficulties intreating hematologic cancers, such as AML. Such cancers are particularlymore intransigent to therapy postremission. Accordingly, there is a needin the art for additional hematologic cancer therapies that target CSC,including to treat AML. The present invention addresses this need bydisclosing a method of treating hematologic cancer using a HIFinhibitor.

SUMMARY OF THE INVENTION

Provided herein is a method for treating a hematologic cancer, which maycomprise administering a HIF inhibitor to a mammal in need thereof. TheHIF inhibitor may be echinomycin, 2-methoxyestradiol, or geldanamycin.The echinomycin may be administered at a non-toxic dose, which may be1-100 mcg/m². The echinomycin may be coadministered with a Hedgehogpathway inhibitor, which may be cyclopamine. The HIF inhibitor may alsobe coadministered with a second cancer therapy.

The hematologic cancer may be a lymphoma or a leukemia, which may beacute myeloid leukemia. The mammal may carry a cytogenetic alteration,which may be 47,XY,+21;46,XY; 45,XX,−7; 46,XY,t(8;2)(q22;q22);49,XX,+8,+8,inv(16)(p13q22),+21; 46,XX.inv(16)(p13q22)/46,XX;46,XY,inv(16)(p13q22); 46,XX,t(2;13)(p23;q12)/46,XX; 45,XY,inv(3)(q21q26.2),−7/46,XY; 47,XY,+4,inv(5)(p15q13)/47,sl,−4,+22;46,XX,t(11;19)(q23;p13.1); 46,XX,t(6;11)(q27;q23)/46,XX; or46,XX,t(1;17)(p13;q25),t(9;1)(p22;q23). The mammal may carry leukemiacells of the phenotype CD38⁻CD34⁺. The patient may carry cancer stemcells, which may be multiple drug resistant, chemoresistant, orradioresistant.

Also provided herein is a method for inducing acute myeloid leukemiaremission, which may comprise administering echinomycin to a patient inneed thereof. The echinomycin may be administered at a non-toxic dose,which may be 1-100 mcg/m². Further provided herein is a method forinhibiting a maintenance or survival function of a cancer stem cell(CSC), which may comprise contacting the CSC with a HIF inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Isolation of CSC from a spontaneous mouse lymphoma. a. Lymphomacells, isolated from an enlarged spleen TGB transgenic mice, were sortedby BD FACSAria into c-Kit⁺Sca-1⁺ or c-Kit⁻Sca-1⁻ fractions. The leftpanel shows tumor phenotype while the right panels show post-sortpurities. b. Colony-forming activity of the tumor cells resides withinthe c-Kit⁺Sca-1⁺ subset. The c-Kit⁺Sca-1⁺ or c-Kit⁻Sca-1⁻ fractions(10³/well) were plated in 1% methylcellulose medium, the colony numberswere counted under a microscope after 7 days of culture. Data shown aremeans and SD of colony numbers in triplicate plates and arerepresentative of three independent experiments. The insert shows themorphology of a typical colony. c. Photograph of spleens from mice thatreceived either c-Kit⁺Sca-1⁺ or c-Kit⁻Sca-1⁻ tumor cells. d. Phenotypicconservation and evolution of lymphoma arising from c-Kit⁺Sca-1⁺ cells.Lymphoma cells were stained with antibodies against CD8 and Vβ8. Theleft panel shows cultured lymphoma cells with predominantly high Vβ8⁺transgenic CD8⁺ cells; while the right panel shows lymphoma cells fromthe spleen of Rag-2^(−/−) mice that received c-Kit⁺Sca-1⁺ cells purifiedfrom cultured lymphoma cells. Note the increase in Vb8-population. Theenlarged double negative populations in the right panel are residentspleen cells.

FIG. 2. HIF addiction of CSC. a. Selective ablation of lymphoma CFU byechinomycin. The cultured lymphoma cells were treated with given dosesof pharmacologically effective drugs in medium for 24 hours. Afterwashing away the drugs, the cells were cultured in 1%methylcellulose-containing medium, and the colony number was countedafter 6 to 7 days. Data shown are means and SD of triplicates and havebeen confirmed by 3 independent experiments. b. A reporter system forHIF activity in the TGB lymphoma. Diagram of lentiviral construct forHIF activity is shown on the top. Pause, a sequence fragment toeliminate the effect of lentiviral promoter; HRE, Hypoxia responseelement (SEQ ID NO: 20); TATA, TATA sequence (TATATAAT) (top). Lowerleft, validation of the reporter. HEK293 cells were transientlytransfected with cDNA encoding mutant HIF1α (P402A/P577A) or vectorcontrol in conjunction with either HRE-driven EGFP reporter, HRE mutantreporter (SEQ ID NO: 21). The cells were analyzed by flow cytometry todetect EGFP expression 36 hours after transduction. Lower right, HIFactivity in the lymphoma CSCs as revealed by co-expression of GFPexpressing cells and c-Kit in WT HRE, but not mutant HRE lentiviralreporters (lower middle panel). c. IC50 of echinomycin in the inhibitionof HIF1α activity in lymphoma CSC. The lymphoma cells transfected withthe HRE reporter system were cultured in the presence of differentconcentration of echinomycin for 12 hours, the % of c-Kit⁺GFP⁺ cells wasnormalized against the untreated group (1.13%, which was defined as100%). The dose that resulted in 50% reduction of the c-Kit⁺GFP⁺ cellsis defined as IC50. d. Selectivity of HIF inhibitor for CFU of lymphomaCSC over the CFU from hematopoietic progenitor cells. c-Kit⁺Sca-1⁺ cellsfrom either TGB or normal bone marrow were treated with givenconcentration of echinomycin overnight prior to plating in the mediumcontaining 1% methylcellulose for CFU assay. The data shown were % ofuntreated controls, and were means+/−S.D. of triplicates. e. Therapeuticeffect of a low dose of echinomycin. Cultured lymphoma cells(1×10⁶/mouse) were injected i.p. into immune competent B10.BR mice.Fourteen days later, 10 μg/Kg/Injection of echimomycin was injected at atwo-day interval for a total of 5 times. Control mice received vehicleonly. The mice were observed daily for survival.

FIG. 3. Increased HIF activity in the lymphoma CSCs under normoxia. a&b. Gene expression. The cultured lymphoma cells were sorted by BDFACSAria sorting system into c-Kit⁺Sca-1⁺ or c-Kit⁻Sca-1⁻ fractions. Thetranscripts of HIF1α, HIF-2α, HIF-3α, VHL and Glut1 were determined byRT-PCR. a. Photograph of RT-PCR products. b. Relative expression asmeasured by real-time PCR: comparison with HPC. Relative expression ofHIF1α and VHL transcripts in FACS sorted c-Kit+Sca-1+ cells from eitherTGB tumor (Spl-tumor) or bone marrow (BM). The expression levels wereexpressed as fractions of house-keeping gene, b-actin. c. Echinomycinselectively induces apoptosis of CSC. The cultured lymphoma cells weretreated with 20 pM echinomycin or vehicle in medium for 16 hours. Thetreated cells were stained with c-Kit and Sca-1, followed by stainingwith Annexin V. The stained cells were analyzed by FACS analysis. Datashown are representative of 3 independent experiments. d. Isolation of 4subsets of tumor cells in AML samples. Bone marrow cells from AMLpatient MI-AML-36 were stained for CD34 and CD38 and sorted into 4subsets for RNA isolation. The presort samples and the gates used forsorting are shown in the left panel and the post sorted populations wereshown in the middle and right panels. The percentages of cells in eachgates are provided in the panels. e. Expression of HIF1α (top) and GLUT1in the subsets. Data shown are means+/−S.D. of transcript levels of thegenes, presented as % of b-actin from the same samples. Enhancedexpression in the CD34⁺CD38⁻ samples have been observed in all 6 AMLsamples tested. f. AML-CFU in all 6 AML samples are highly sensitive toechinomycin. AML samples (2.5×10⁵/ml) from either peripheral blood (PB)or bone marrow (BM) were pretreated with given concentrations ofechinomycin in 2 ml medium for 24 hours. Viable treated cells were thenplated at 105/well for CFU assay in triplicates. The colony numbers werecounted 7-10 days later. The data shown were % means+/−S.D. of untreatedcontrols.

FIG. 4. ShRNA silencing revealed a critical role for HIF1α in CSCmaintenance. a. Silencing HIF1α abrogates the c-Kit⁺Sca-1⁺ CSC. TGBtumor cells were infected with either lentiviral vector control withscrambled ShRNA (core sequence 5′-tct cgt cat aac aag ttg a-3), orlentiviral vector expressing two independent ShRNA (sh-1 or sh-2). Threedays after infection, the bulk tumor cells were analyzed by flowcytometry. The GFPhi and GFPlo cells were gated and analyzed forexpression of c-Kit and Sca-1. b. HIF1α shRNA reduces CFU. The culturedlymphoma cells were infected with either lentiviral HIF1α shRNA orvector with scrambled ShRNA by spinoculation, and the infected cellswere selected with 5 μg/ml of blasticidin for one week. The infectedcells were seeded into 1% of methylcellulose culture medium at thedensity of 2×10⁵/well. The colony numbers were counted under amicroscope. Data shown are means and SD of colony numbers in triplicatesand are representative of three independent experiments. c. HIF1α shRNAsabrogate tumor-initiating activity. TGB tumor cells were infected 3times with lentiviral expressing scrambled ShRNA or HIF1α ShRNA and theninjected into B10.BR mice (9×10⁵/mouse, i.p.). The survival of therecipient mice (n=5) was compared by Kaplan-Meier analysis. All micethat succumbed have developed lymphoma as revealed by necropsy. All datain this figure have been repeated at least twice.

FIG. 5. Down-regulation of the Vhl gene is essential for maintenance ofCSC. a. Down-regulation of Vhl transcript in c-Kit⁺Sca-1⁺ cells. TGBthymoma cells were sorted into c-Kit+Sca-1+ and c-Kit⁻Sca-1⁻ subsets, asdescribed in FIG. 1, the levels of Vhl transcripts were determined byreal-time PCR. b. Ectopic expression of Vhl ablated CSC. TGB tumor cellswere infected with either lentiviral vector control, or lentiviralvector expressing two Vhl cDNA. Three days after infection, the bulktumor cells were analyzed by flow cytometry. The GFPhi and GFPlo cellswere gated and analyzed for expression of c-Kit and Sca-1. c. Vhlexpression reduces tumor CFU. The cultured lymphoma cells were infectedwith either lentiviral Vhl cDNA or vector by spinoculation, and theinfected cells were selected with 5 μg/ml of blasticidin for one week.The transduced cells were seeded into 1% of methylcellulose culturemedium at a density of 2×10⁵/well. The colony numbers were counted undera microscope. Data shown are means and SD of colony numbers intriplicates and are representative of three independent experiments. d.Ectopic expression of Vhl cDNA inhibits tumor-initiating activity. TGBtumor cells were infected 3 times with lentiviral expressing vectoralone or HIF1α ShRNA and then injected into B10.BR mice (9×10⁵/mouse,i.p.). The survival of the recipient mice (n=5) was compared byKaplan-Meier analysis. The development of lymphoma was confirmed bynecropsy of the succumbed mice. This experiment has been repeated twice.

FIG. 6. HIF works in concert with Notch pathway to maintain CSC. a.Inhibition of colony-forming activity of CSC. The cultured lymphomacells were treated with given doses of L685, 458 for 24 hours. Afterthat, the cells were cultured in 1% methylcellulose-containing medium,and the colony number was counted after 6 to 7 days. Data shown aremeans+/−SD of triplicate samples and are representative of those of atleast 3 independent experiments. b. Enhanced Notch activity in CSC, asindicated by the levels of Hes1 transcripts. The lymphoma cells fromspleen with tumor in TGB transgenic mouse were sorted into c-kit⁺Sca-1⁺or c-kit⁻Sca-1⁻ fractions. The expressions of Hes1 and mRNA in these twofractions were measured by real time PCR. c. Inhibition of Notchactivity by 3 distinct HIF inhibitors. Cultured TGB lymphoma cells werestained with APC conjugated anti-c-Kit-antibody and enriched twice usinganti-APC coated MACS beads according to manufacturer's protocol(Militenyi Biotec). The c-kit positive cells-enriched samples (60.4%c-Kit⁺ cells) were treated with inhibitors of HIF for 16 hours. The mRNAfrom the treated cells were extracted for quantitation by real-time PCR.Data shown are means+/−SD of triplicates and represent those from atleast 3 independent experiments. 2ME2, 2-methoxyestradiol; GA,geldamycin. d-g. A critical role for Notch in maintenance of CSC, asrevealed by ectopic expression of Notch I-C dRdA1-42dOP. d. A truncatedNotch gene with potent dominant negative activity in inhibiting theexpression of Notch target gene Hes. The upper left panel shows thediagram of the intracellular portion of Notch protein, with the positionof RAM, 7 ankyrin repeats (ANK1-7), transcriptional activation domain(TAD), C-terminal OPA (O) and PEST (P) sequence are marked. The lowerleft panel showed the composition of the dRdA1-4dOP mutant lacking RAM,ANK1-4 and C-terminal O and P sequence, but with insertion of nuclearlocalization sequence (NLS). The right panel show dominant inhibition ofHes expression. After three consecutive transductions by either vectorcontrol or the dRdA1-4dOP mutant, the RNA were isolated and thetranscripts of Hes measured by quantitative PCR. Data shown are means oftriplicates and have been reproduced by two independent experiments. e.Notch I-C deltaRAM abrogates the c-Kit⁺Sca-1⁺ subset. TGB tumor cellswere infected with either lentiviral vector control, or lentiviralvector expressing dRdA1-4dOP. Three days after infection, the bulk tumorcells were analyzed by flow cytometry. The GFPhi and GFPlo cells weregated and analyzed for expression of c-Kit and Sca-1. f. NotchIC-dRdA1-42dOP reduces in vitro self-renewal activity of CSC. Thecultured lymphoma cells were infected with either lentiviral vectorencoding dRdA1-4dOP or vector control by spinoculation. The same numbersof infected cells were seeded into 1% of methylcellulose culture mediumfor 3 days and the numbers of colony with GFP were counted undermicroscope. The same procedure was performed for second round colonyformation assay. Data shown are the means and SD of the colony numbersin triplicate plates, and are representative of those from at leastthree independent experiments. g. dRdA1-4dOP abrogates tumor-initiatingactivity. TGB tumor cells were infected 3 times with lentiviralexpressing vector alone or HIF1α ShRNA and then injected into B10.BRmice (1×10⁶/mouse, i.p.). The survival of the recipient mice wascompared by Kaplan-Meier analysis with statistical significancedetermined by log-rank tests. All succumbed mice have developed lymphomaas revealed by necropsy. This experiment has been repeated twice. h-l.HIF1α inhibits negative feedback regulation of Hes1 by preventing Hes1binding to the N-boxes in the Hes1 promoter. h. Diagram of Hes1promoter. Detail sequence is provided in FIG. 16. i. HIF1α did notco-operate with Notch directly in activating Hes1 promoter. The Hes1promoter sequence (−225 to +65, TSS as +1) were linked to GFP andtransfected into 293 cells in conjunction with vector controls, orvector containing cDNA encoding HIF1α (P402, 577>A, called HIF1α-PA),Notch-IC cDNA or Notch-IC+ HIF1αPA. The promoter activity is measured bythe green fluorescence intensity of transfected cells. Data shown wererelative intensities. The intensity of Hes1-GFP reporter is defined as1.0. Transfection efficiency is normalized by co-transfected Renillaluciferase. j. HIF1α partially inhibited Hes1-mediated repression of theHes1 promoter. As in i, except that the Hes1 or mutant HIF1α cDNA areused. k. HIF1α diminishes the negative auto-regulation of Hes1expression in Notch signaling. As in i, except different combination ofcDNAs were used. Activity of Hes1 reporter in the absence of transfectedHes. HIF1α-PA and Notch is defined as 1.0. l. Competitive inhibitionbetween HIF1α-PA and Hes1 to Hes promoter, as revealed by chIP. cDNAsencoding Flag or Myc-tagged Hes1 and HIF-1αPA were transfected into 293cells. Thirty-six hours after transfection, the transfectants weresubject or ChIP. Equal fractions of cells in each group were used forWestern blot to confirm essentially identical levels of proteinexpression when Hes1 and HIF1α-PA were transfected alone or incombination (data not shown). The data present are means+/−S.D. (n=3) of% of input DNA, as measured by real-time PCR using primers marked inFIG. 16. Data shown in i-l are means+/−S.D. of triplicates. Theexperiments have been repeated at least 3 times.

FIG. 7. The progenies of c-Kit+Sca-1+ subset are heterogenous with asmall fraction retaining the c-Kit+Sca-1+ phenotype. Ex vivo tumor cellswere sorted as described in FIG. 1a and plated in FIG. 1b . Five dayslater, the cells from the colonies were re-analyzed for expression ofc-Kit and Sca-1.

FIG. 8. Cells with HIF activity express both c-Kit and Sca-1. TGB tumorcells were infected with lentiviral vector containing the WT HREsequence as depicted in FIG. 2d . Three days after infection, the tumorcells were stained with anti-c-Kit and anti-Sca-1 mAbs. The GFP⁺ andGFP⁻ cells were analyzed for the expression of c-Kit and Sca-1.

FIG. 9. Inhibition of colony formation of c-Kit⁺Sca-1⁺ tumor cells byHIF-1α inhibitors. Data shown were means and S.D. of triplicate cultureand have been repeated at least 3 times. a. Continuous inhibition byechinomycin. c-Kit⁺Sca-1⁺ cells sorted from TGB tumor cells werecultured and resorted for c-Kit⁺Sca-1⁺ cells. The first and secondrounds of sorted cells (1000/well) were cultured in the presence orabsence of different doses of echinomycin for 24 hours. The drugs werewashed away and the cells plated. The colonies were counted 5 days afterculture. b. Inhibition by other classes of HIF1α inhibitors,2-Methoxyestradiol (2ME2) and Geldanamycin (GA), but not by a P38inhibitor, SB203580. As described in a, except additional drugs wereused as control. c. At low concentrations (5-20 pM), echinomycininhibits self-renewal of colony-initiating cells without affectingcolony formation. Equal aliquots of TGB tumor cells were cultured inmethylcellulose-containing medium in the presence of givenconcentrations of echinomycin for 5 days when the first round colonieswere counted. After washing away the drugs, Equal aliquots of cells fromthe first round were replated. The newly formed colonies were counted.The numbers of colonies were normalized against the untreated group. Thenumbers of colonies in untreated cultures: first round, 362; secondround 202.

FIG. 10. Continuous resistance of HPC activity to high doses ofechinomycin. For the first round, total bone marrow cells (106) weretreated with given concentration of echinomycin for 24 hours. Afterwashing away the drugs, aliquots corresponding to 0.25×10⁶ seededcells/well were plated into methylcellulose containing medium for CFUassay. The colonies were counted on day 5. For the second roundanalysis, 0.5×10⁶ viable cells isolated from the untreated group in thefirst round were incubated with given concentration of echinomycin.Aliquots corresponding to 5×10⁴ cells/well were replated and thecolonies were counted on day 5. The data shown are means and SD oftriplicates.

FIG. 11. Therapeutic effect of echinomycin administrated 4 days aftertumor transplantation. Cultured lymphoma cells (1×10/mouse) weretransplanted into immune competent B10.BR mice i.p. Four days later, 10μg/Kg/Injection of echimomycin was injected with a two day interval fora total of 3 times. Control mice received vehicle only.

FIG. 12. Multiple HIF-1α inhibitors repress self-renewal and tumorinitiating activity of the TGB tumor cells. a. Dose-dependent inhibitionof colony formation by 2-methoxyestradiol (2ME2) and Geldanamycin. b.Inhibition of tumor formation by HIF inhibitors 2ME2 and Geldanamycin.Cultured lymphoma cells (9×10⁵/mouse) were injected into immunecompetent B10.BR mice i.p. Eight days later, given doses of 2ME2 andGeldanamycin were injected with a two day interval for a total of 5times. Control mice received vehicle only. The mice were observed dailyfor survival. The P values given were obtained by log-rank tests incomparison to treated and control groups.

FIG. 13. At ranges used in the study, Echinomycin inhibits HIF1α but notcMyc activity. HEK293 cells were co-transfected with the Ebox or HREreporters (see FIG. 2b ) with the vector encoding c-Myc or HIF1α-PA.After 16 hours, the cells were treated with different concentrations ofEchinomycin for additional 24 hrs. The transcriptional activities ofc-Myc and HIF1α were quantified by flow cytometry. The untreated groupis defined as 100%. Data shown are means+/−S.D. of triplicates. Theexperiment was repeated three times.

FIG. 14. Verification of the efficacy of HIF-1α shRNA. The 293T cellswere transfected with a cDNA encoding the HIF-1α mutant (P402A/P577A)that is stable under normoxia condition in conjunction with eithercontrol vectors V (Core scrambled sequence: 5′-cgcgtagcgaagctca taa-3′)or HIF-1α shRNA-1 and -2. The cell lysates were probed with anti-FlagmAb 24 hours after transfection.

FIG. 15. TGB tumor cells express both Notch 1 and Notch 2. The TGB tumorcells were sorted into c-Kit+Sca-1+ and c-Kit−Sca-1− subsets. Theexpression of Notch family members were detected by RT-PCR.

FIG. 16. Sequence of mouse (SEQ ID NO: 22) and human (SEQ ID NO: 23)Hes1 promoter sequence. The N-box, CBF1, TSS, and HRE are labeled. Theprimers used for reporter construction and real-time PCR in ChIP assaysare also marked.

FIG. 17. Echinomycin abrogated AML in NOD-SCID mice. A. Elimination ofAML-derived human cells in the blood of recipient mice. Data shown aremeans and S.D. (n=3). B. Lack of human cells in echinomycin-treatedmice. Data shown are representative profile of human CD45 expressionamong bone marrow cells. Essentially identical profiles were obtainedwith two other mice. B. Human AML cells in the bone marrow of untreatedmice. The left panel shows human CD45 staining of bone marrow cells,while the right panels show phenotypes of the gated human CD45⁺ cells.Data shown are representative of three mice per group.

FIG. 18. HIF1α is a target for therapeutic elimination of human AML in axenogeneic mouse model. a. Isolation of 4 subsets of tumor cells in AMLsamples. Bone marrow cells from AML patient MI-AML-71 were stained forCD34 and CD38 and sorted into 4 subsets for RNA isolation. The presortsamples and the gates used for sorting are shown in the left panel andthe post-sorted populations are shown in the middle and right panels.The percentages of cells in each gate are provided in the panels. b.Expression of HIF1α (top) and GLUT1 in the subsets. Data shown aremeans+/−S.D. of transcript levels of the genes, presented as % ofβ-actin from the same samples. Enhanced expression in the CD34⁺CD38⁻samples was observed in all 6 AML samples tested. c. AML-CFU in all 6AML samples were highly sensitive to echinomycin. AML samples(2.5×10⁵/ml) from either peripheral blood (PB) or bone marrow (BM) werepretreated with given concentrations of echinomycin in 2 ml medium for24 hours. Treated viable cells were then plated at 10⁵/well for CFUassay in triplicates. The colony numbers were counted 7-10 days later.The data shown are % means+/−S.D. of untreated controls. d. Echinomycinselectively eliminates the CD34⁺CD38⁻ subset of AML cells. Primary AMLsamples were cultured with given doses of echinomycin or vehicle controlfor 30 hours in RPMI 1640 containing 10% fetal calf serum and humancytokine cocktail consisting of CSF, GM-CSF and IL-3 at a density of5×10⁵/ml. The cells were stained with antibodies against CD34, CD38 inconjunction with Annexin V and DAPI. Data shown are the % of AnnexinV⁺DAPI^(−/+) cells. The Annexin V⁺ cells % in vehicle treated group wassubtracted. The filled symbols show the data for the CD34⁺CD38⁻ subsets,while the open symbols show data for the bulk leukemia cells (CD34⁺CD38⁺for AML9, AML32, AML60 and AML71 and CD34⁺CD38⁺ for AML15 and AML36).These data were repeated twice. e-h. Therapeutic effect of echinomycin.e. Therapeutic effect of human AML in NOD-SCID mice, data shown are % ofhuman CD45 (hCD45)⁺ cells in the bone marrow of the recipient mice at 40days after last treatment. The therapeutic effect has been repeatedtwice. f. Echinomycin does not induce differentiation of AML cells invivo, as revealed by lack of mature myeloid markers on the bulk of humancells in treated and untreated group. Data shown are profiles of CD14and CD15 among human CD45⁺ cells. g. Selective depletion of theCD34⁺CD38⁻ subset by echinomycin. Data shown in the top panels are theabundance of CD34⁺CD38⁻ subsets in mouse bone marrow, while the lowerpanel shows that within human leukemia cells. h. Despite presence ofleukemia cells, bone marrow from echinomycin-treated mice failed toreinitiate leukemia in the new NOD-SCID mice. Representative profilesare presented in the top panel, while the summary data from 5 mice pergroup are presented in the lower panel.

DETAILED DESCRIPTION

The inventors have made the surprising discovery that the HIF inhibitorechinomycin is capable of treating a hematologic cancer at a non-toxicdose. This indicates a unique susceptibility of lymphoma CSC toechinomycin, and that as little as 30 mcg/m² of echinomycin issufficient to abrogate lymphoma in 100% of the recipients. For example,in vitro, AML-CFUs of all 6 cases of human AML samples tested werehighly susceptible to echinomycin.

Echinomycin was brought into clinical trials about 20 years ago based onits antitumor activity against two i.p. implanted murine tumors, the B16melanoma and the P388 leukemia. However, testing of the efficacy ofechinomycin phase 11 clinical trials for a number of solid tumorsrevealed that echinomycin exhibits significant toxicity, and had minimalor no efficacy. The efficacy of echinomycin for treating hematologicalcancer had not been tested. Additionally, the body-surface adjusted doseused in previous human clinical trials was about 100-fold higher thanthe dose the inventors have discovered to be effective for treatinghematologic cancer. The toxicity observed in previous human trials waslikely due to excess dose used, while the lack of effect may have beendue to clinical endpoints that do not reflect the unique requirement forHIF in lymphoma CSC. Thus, echinomycin may be used to treat hematologiccancer associated with CSC, including lymphoma, and in particular, at anon-toxic dose.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

A “peptide” or “polypeptide” is a linked sequence of amino acids and maybe natural, synthetic, or a modification or combination of natural andsynthetic.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

2. HYPOXIA-INDUCIBLE FACTOR INHIBITOR

Provided herein is an inhibitor of Hypoxia-Inducible Factor protein(HIF). The HIF inhibitor may be echinomycin, 2-methoxyestradiol, orgeldanamycin.

a. Echinomycin

The echinomycin may be a peptide antibiotic such asN,N′-(2,4,12,15,17,25-hexamethyl-11,24-bis(1-methylethyl)-27-(methylthio)-3,6,10,13,16,19,23,26-octaoxo-9,22-dioxa-28-thia-2,5,12,15,18,25-hexaazabicyclo(12.12.3)nonacosane-7,20-diyl)bis(2-quinoxalinecarboxamide).The echinomycin may be a microbially-derived quinoxaline antibiotic,which may be produced by Streptomyces echinatus. The echinomycin mayhave the following structure.

The echinomycin may have a structure as disclosed in U.S. Pat. No.5,643,871, the contents of which are incorporated herein by reference.The echinomycin may also be an echinomycin derivative, which maycomprise a modification as described in Gauvreau et al., Can JMicrobiol, 1984; 30(6):730-8; Baily et al., Anticancer Drug Des 1999;14(3):291-303; or Park and Kim, Bioorganic & Medicinal ChemistryLetters, 1998; 8(7):731-4, the contents of which are incorporated byreference. The echinomycin may also be a bis-quinoxaline analog ofechinomycin

b. HIF

The HIF may be a functional hypoxia-inducible factor, which may comprisea constitutive b subset and an oxygen-regulated a subunit. The HIF maybe over-expressed in a broad range of human cancer types, which may be abreast, prostate, lung, bladder, pancreatic or ovarian cancer. While notbeing bound by theory, the increased HIF expression may be a directconsequence of hypoxia within a tumor mass. Both genetic andenvironmental factors may lead to the increased HIF expression evenunder the normoxia condition. Germline mutation of the von Hippel-Lindaugene (VHL), which may be the tumor suppressor for renal cancer, mayprevent degradation HIF under normoxia. It may be possible to maintainconstitutively HIF activity under normoxia by either upregulation of HIFand/or down regulation of VHL. The HIF may be HIF1α or HIF2α.

c. Pharmaceutical Composition

Also provided is a pharmaceutical composition comprising the HIFinhibitor. The pharmaceutical composition may be in the form of tabletsor lozenges formulated in a conventional manner. For example, tabletsand capsules for oral administration may contain conventional excipientsmay be binding agents, fillers, lubricants, disintegrants and wettingagents. Binding agents include, but are not limited to, syrup, accacia,gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers may be lactose, sugar, microcrystallinecellulose, maizestarch, calcium phosphate, and sorbitol. Lubricantsinclude, but are not limited to, magnesium stearate, stearic acid, talc,polyethylene glycol, and silica. Disintegrants may be potato starch andsodium starch glycollate. Wetting agents may be sodium lauryl sulfate.Tablets may be coated according to methods well known in the art.

The pharmaceutical composition may also be liquid formulations such asaqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.The pharmaceutical composition may also be formulated as a dry productfor constitution with water or other suitable vehicle before use. Suchliquid preparations may contain additives such as suspending agents,emulsifying agents, nonaqueous vehicles and preservatives. Suspendingagents may be sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents may belecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles may beedible oils, almond oil, fractionated coconut oil, oily esters,propylene glycol, and ethyl alcohol. Preservatives may be methyl orpropyl p-hydroxybenzoate and sorbic acid.

The pharmaceutical composition may also be formulated as suppositories,which may contain suppository bases such as cocoa butter or glycerides.The pharmaceutical composition may also be formulated for inhalation,which may be in a form such as a solution, suspension, or emulsion thatmay be administered as a dry powder or in the form of an aerosol using apropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Agents provided herein may also be formulated as transdermalformulations comprising aqueous or nonaqueous vehicles such as creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

The pharmaceutical composition may also be formulated for parenteraladministration such as by injection, intratumor injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The pharmaceutical composition mayalso be provided in a powder form for reconstitution with a suitablevehicle including, but not limited to, sterile, pyrogen-free water.

The pharmaceutical composition may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The pharmaceutical composition may beformulated with suitable polymeric or hydrophobic materials (as anemulsion in an acceptable oil, for example), ion exchange resins, or assparingly soluble derivatives (as a sparingly soluble salt, forexample).

(1) Administration

Administration of the pharmaceutical composition may be orally,parenterally, sublingually, transdermally, rectally, transmucosally,topically, via inhalation, via buccal administration, or combinationsthereof. Parenteral administration includes, but is not limited to,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, and intraarticular. For veterinary use, theagent may be administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal. The pharmaceutical composition maybe administered to a human patient, cat, dog, large animal, or an avian.

The pharmaceutical composition may be administered simultaneously ormetronomically with other treatments. The term “simultaneous” or“simultaneously” as used herein, means that the pharmaceuticalcomposition and other treatment be administered within 48 hours,preferably 24 hours, more preferably 12 hours, yet more preferably 6hours, and most preferably 3 hours or less, of each other. The term“metronomically” as used herein means the administration of the agent attimes different from the other treatment and at a certain frequencyrelative to repeat administration.

The pharmaceutical composition may be administered at any point prior toanother treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr,92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins, 10mins, 9 mins, 8 mins, 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2mins, and 1 mins. The pharmaceutical composition may be administered atany point prior to a second treatment of the pharmaceutical compositionincluding about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9 mins., 8mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.

The pharmaceutical composition may be administered at any point afteranother treatment including about 1 min, 2 mins., 3 mins., 4 mins., 5mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins.,25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60hr, 62 hr, 64 hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80hr, 82 hr, 84 hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100hr, 102 hr, 104 hr, 106 hr, 108 hr, 110 hr, 112 hr. 114 hr, 116 hr, 118hr, and 120 hr. The pharmaceutical composition may be administered atany point prior after a pharmaceutical composition treatment of theagent including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr,108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins. and1 mins.

d. Dosage

The pharmaceutical composition may be administered in a therapeuticallyeffective amount of the HIF inhibitor to a mammal in need thereof. Thetherapeutically effective amount required for use in therapy varies withthe nature of the condition being treated, the length of time desired toinhibit HIF activity, and the age/condition of the patient.

The dose may be a non-toxic dose. The dose may also be one at which HIFactivity is inhibited, but at which c-Myc activity is unaffected. Ingeneral, however, doses employed for adult human treatment typically maybe in the range of 1-100 mcg/m² per day, or at a threshold amount of1-100 mcg/m² per day or less, as measured by a body-surface adjusteddose. The desired dose may be conveniently administered in a singledose, or as multiple doses administered at appropriate intervals, forexample as two, three, four or more sub-doses per day. Multiple dosesmay be desired, or required.

The dosage may be a dosage such as about 1 mcg/m², 2 mcg/m, 3 mcg/m², 4mcg/m², 5 mcg/m², 6 mcg/m², 7 mcg/m², 8 mcg/m², 9 mcg/m², 10 mcg/m², 15mcg/m², 20 mcg/m², 25 mcg/m², 30 mcg/m², 35 mcg/m², 40 mcg/m², 45mcg/m², 50 mcg/m², 55 mcg/m², 60 mcg/m², 70 mcg/m², 80 mcg/m², 90mcg/m², 100 mcg/m², 200 mcg/m², 300 mcg/m², 400 mcg/m, 500 mcg/m², 600mcg/m², 700 mcg/m², 800 mcg/m², 900 mcg/m², 1000 mcg/m², 1100 mcg/m, or1200 mcg/m², and ranges thereof.

The dosage may also be a dosage less than or equal to about 1 mcg/m², 2mcg/m², 3 mcg/m², 4 mcg/m², 5 mcg/m², 6 mcg/m², 7 mcg/m², 8 mcg/m², 9mcg/m², 10 mcg/m², 15 mcg/m², 20 mcg/m², 25 mcg/m², 30 mcg/m², 35mcg/m², 40 mcg/m², 45 mcg/m², 50 mcg/m², 55 mcg/m², 60 mcg/m², 70mcg/m², 80 mcg/m², 90 mcg/m², 100 mcg/m², 200 mcg/m², 300 mcg/m², 400mcg/m², 500 mcg/m², 600 mcg/m², 700 mcg/m², 800 mcg/m², 900 mcg/m², 1000mcg/m², 1100 mcg/m², or 1200 mcg/m², and ranges thereof.

e. Coadministration

The HIF inhibitor may be coadministered with another pharmacologicalagent. The agent may be an inhibitor of the Hedgehog pathway, which maybe cyclopamine. The agent may also be a second cancer therapy. Thecancer therapy may be a cytotoxic agent or cytostatic agent. Thecytotoxic agent may be selected from the group consisting of: alkylatingagents (including, without limitation, nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas and triazenes): uracilmustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide,melphalan, chlorambucil, pipobroman, triethylene-melamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, and temozolomide; antimetabolites (including,without limitation, folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors): methotrexate,5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine;natural products and their derivatives (for example, vinca alkaloids,antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins):vinblastine, vincristine, vindesine, bleomycin, dactinomycin,daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel(paclitaxel is commercially available as Taxol®), mithramycin,deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (preferablyIFN-{umlaut over (γ)}), etoposide, and teniposide. Other proliferativecytotoxic agents are navelbene, CPT-11, anastrazole, letrazole,capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

The cytotoxic agent may be a microtubule affecting agent, which mayinterfere with cellular mitosis. The microtubule affecting agent may beselected from the group consisting of: allocolchicine (NSC 406042),halichondrin B (NSC 609395), colchicine (NSC 757), colchicinederivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine(NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®, NSC 125973),Taxol® derivatives (e.g., derivatives (e.g., NSC 608832), thiocolchicine(NSC 361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC49842), vincristine sulfate (NSC 67574), natural and syntheticepothilones including but not limited to epothilone A, epothilone B, anddiscodermolide (see Service, (1996) Science, 274:2009) estramustine,nocodazole, and MAP4. The microtubule affecting agent may also be asdescribed in Bulinski (1997) J. Cell Sci. 110:3055 3064; Panda (1997)Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer Res.57:3344-3346; Nicolaou (1997) Nature 387:268-272; Vasquez (1997) Mol.Biol. Cell. 8:973-985; and Panda (1996) J. Biol. Chem 271:29807-29812,the contents of which are incorporated herein by reference.

The cytotoxic agent may also be selected from the group consisting of:epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes such ascis-platin and carboplatin; biological response modifiers; growthinhibitors; antihormonal therapeutic agents; leucovorin; tegafur; andhaematopoietic growth factors.

The cytostatic agent may be selected from the group consisting of:hormones and steroids (including synthetic analogs):17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone,fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, and zoladex. The cytostatic agent may also be anantiangiogenic such as a matrix metalloproteinase inhibitor or a VEGFinhibitor, which may be an anti-VEGF antibody or small molecule such asZD6474 or SU6668. The agent may also be an Anti-Her2 antibody fromGenentech, an EGFR inhibitor such as EKB-569 (an irreversibleinhibitor), or an Imclone antibody C225 immunospecific for the EGFR, ora src inhibitor.

The cytostatic agent may also be selected from the group consisting of:Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependentcarcinomas non-proliferative; the antiestrogen Tamoxifen® which inhibitsthe proliferation or growth of estrogen dependent breast cancer; and aninhibitor of the transduction of cellular proliferative signals. Theinhibitor of the transduction of cellular proliferative signals may beselected from the group consisting of epidermal growth factorinhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinaseinhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.

3. METHOD

a. Treating a Hematologic Cancer

Provided herein is a method of treating a hematologic cancer. The methodmay comprise administering a HIF inhibitor to a mammal in need thereof.The mammal may be a human patient. The hematologic cancer may belymphoma or leukemia. The hematologic cancer may be treated byinhibiting a maintenance or survival function of a CSC. Without beingbound by theory inhibiting HIF may target both the cancer stem cell andcancer resistance.

Further without being bound by theory, the CSC in the hematologic cancermay require self-renewal, which may be similar to the requirement intissue cells. The CSC may require a hypoxic environment, and exposure toa high level of oxygen may reduce CSC function. Self-renewal of CSCfunction may be strongly inhibited by drugs targeting the HIF pathway.CSC may be addicted to the HIF, which may be associated withover-expression of HIF and down-regulation of VHL. HIF over-expressionand VHL down-regulation may be critical in the maintenance of CSC. HIFmay work in concert with the Notch pathway to mediate self-renewal ofthe lymphoma CSC.

(1) Cancer Stem Cell

The cancer stem cell may be chemoresistant or radioresistant. The CSCmay also be multiple drug resistant.

(2) Acute Myeloid Leukemia

The leukemia may be acute myeloid leukemia (AML). The AML may beassociated with a CSC characterized by the genotype CD38⁻CD34⁺. The AMLmay also be associated with a patient who carries a cytogeneticalteration. The cytogenetic alteration may be selected from the groupconsisting of: 47,XY,+21;46,XY; 45,XX,−7; 46,XY,t(8;21)(q22;q22);49,XX,+8,+8,inv(16)(p13.1q22),+21; 46,XX,inv(16)(p13q22)/46,XX;46,XY,inv(16)(p13q22); 46,XX,t(2;13)(p23;q12)/46,XX;45,XY,inv(3)(q21q26.2),−7/46,XY; 47,XY,+4,inv(5)(p15q13)/47,sl,−4,+22;46,XX,t(11;19)(q23;p13.1); 46,XX,t(6;11)(q27;q23)/46,XX; and46,XX,t(1;17)(p13;q25),t(9;11)(p22;q23).

The CSC of the AML may be extremely sensitive to the HIF inhibitor. TheCFU of AML may be highly susceptible to the HIF inhibitor, with an IC50between 50-120 pM. The HIF inhibitor may be used to eliminate CSC of AMLas part of postremission therapy.

b. Inducing Acute Myeloid Leukemia Remission

Also provided herein is a method for inducing remission of acute myeloidleukemia, which may comprise administering the HIF inhibitor to a mammalin need thereof. The HIF inhibitor may be administered to the mammalduring remission of acute myeloid leukemia to prevent future relapse.The HIF inhibitor may be administered as elsewhere disclosed herein.

c. Inhibiting a Maintenance or Survival Function of a CSC

Further provided herein is a method for inhibiting a maintenance orsurvival function of a CSC. Contacting the CSC with the HIF inhibitormay inhibit the maintenance or survival function. The contacting maycomprise administering the HIF inhibitor to a mammal in need ofinhibiting the maintenance or survival function of the CSC. The HIFinhibitor may be administered as elsewhere disclosed herein.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 Identification of Self-Renewing Lymphoma Initiating Cells inSyngeneic Immune Competent Host

Hundred percent of the transgenic mice (TGB) with insertional mutationof the Epm2a gene succumb to lymphoma. In search for the expression ofpotential stem cell markers in the TGB lymphoma cells, it was found thata small subset of cells expressed both c-Kit and Sca-1, which partlyconstitute markers for HSC (FIG. 1a ). To test if these cells may haveCSC activity, lymphoma cells from the spleen of TGB transgenic mice withtumors were sorted based on expression of both c-Kit and Sca-1 (FIG. 1aright panels). As shown in FIG. 1b , no colony formed from 10³ ofc-Kit⁻Sca-1⁻ cells. In striking contrast, 10³ of cells with c-Kit⁺Sca-1⁺yielded about 800 colonies, which suggests that every cells in thesubset was CFU. Most colonies are tightly packed (FIG. 1b insert), incontrast to the diverse colonies obtained from bone marrow HSC. Theprogenies of the c-Kit⁺Sca-1⁺ cells consist of both c-Kit⁺Sca-1⁺ andc-Kit⁺Sca-1⁺ subsets (FIG. 7). Therefore, the c-Kit⁺Sca-1⁺ cells are theself-renewing population among the single cells isolated from the TGBlymphoma. To determine if the c-Kit⁺Sca-1⁺ cells are also thelymphoma-initiating cells in vivo, we injected either c-Kit⁺Sca-1⁺ cellsor c-Kit⁻Sca-1⁺ cells intraperitoneally (i.p.) into the syngeneic B10BRmice. As shown in Table 1, expt 1, three out of three mice receiving 100c-Kit⁺Sca-1⁺ cells developed lymphomas usually within 10 weeks afterinjection, yet none of the recipients of 10⁴ of c-Kit−Sca-1− cells dideven after 40 weeks of observation. Similar results were obtained whenthe experiments were repeated by intravenous injection (Table 1, Expt2). The lymphomas are characterized by enlarged spleens (FIG. 1c ),lymph nodes, and metastasis to the liver and lung but not thymus (datanot shown), unlike the spontaneously developed lymphoma that firstappeared as thymoma and then metastasized into other organ. Furthermore,no constitution of other cell lineages was achieved from thec-Kit⁺Sca-1⁺ subset isolated from tumors, which indicates that thec-Kit⁺Sca-1⁺ cells are not the tumor-infiltrating HSC. In three roundsof serial transplantation (Table 1, expt 5), the c-Kit⁺Sca-1⁺ cells, butnot the c-Kit⁻Sca-1⁻ cells, gave rise to lymphoma at a comparablepotency. The tumors maintained expression of T-cell marker CD8, butgradually lost cell surface expression of the transgenic TCR (FIG. 1dand Table S1). More importantly, the frequency of the c-Kit⁺Sca-1⁺ cellsremained around 1% (Table S1). Thus, the self-renewing tumor-initiatingcells are among the c-Kit⁺Sca-1⁺ tumor cells.

TABLE 1 Identification of CSC using c-Kit and Sca-1 markers Number ofcells injected Expt Donor Recipient 10,000 1,000 500 100 1. c-Kit⁺Sca-1⁺B10.BR — — 3/3 3/3 c-Kit⁻Sca-1⁻ B10.BR 0/3 — — — 2. c-Kit⁺Sca-1⁺ B10.BR— — 4/4 3/3 c-Kit⁻Sca-1⁻ B10.BR 0/3 — 0/3 — 3. c-Kit⁺Sca-1⁺ B10.BR — —5/5 5/5 c-Kit⁻Sca-1⁻ B10.BR 1/5 0/5 — — 4. c-Kit⁺Sca-1⁺ RAG2^(−/−) — —5/5 5/5 c-Kit⁻Sca-1⁻ RAG2^(−/−) 1/5 0/5 — — 5. Serial transplantationRound 1 c-Kit⁺Sca-1⁺ B10.BR — — 3/3 3/3 c-Kit⁻Sca-1⁻ B10.BR 1/3 0/3 — —Round 2 c-Kit⁺Sca-1⁺ B10.BR — — — 5/5 c-Kit⁻Sca-1⁻ B10.BR 2/5 — — —Round 3 c-Kit⁺Sca-1⁺ B10.BR — — — 3/3 c-Kit⁻Sca-1⁻ B10.BR 0/3(5000/mouse) — —

The donor cells were isolated from either ex vivo lymphoma (expt 1 and2) or those that have been cultured for more than 30 passages in vitro.The routes of injection were intraperitoneal (i.p.) for experiments 1,3, and 4, and intravenous for experiment 2. There was no tumor growth(0/3) when 10 c-Kit⁺Sca-1⁺ cells were transplanted into B10.BR mice. Inexperiment 5, donor cells were isolated from ex vivo lymphoma andinjected i.p. The lymphoma cells obtained in round 1 were sorted andinjected for the second around, then repeated for the third round. Thetumor-free mice were observed for 22-40 weeks to confirm the lack oftumor growth.

TABLE S1 % marker⁺ cells Markers Primary Round 1 Round 2 Round 3c-Kit⁺-Sca-1 3.80 3.92 4.81 1.32 c-Kit⁻Sca-1⁺ 1.52 15.38 19.5 5.14c-Kit⁺Sca1⁺ 0.87 0.81 0.97 0.83 CD8⁺Vβ8⁻ 3.48 ND 47.39 54.27 CD8⁺Vβ8⁻92.36 ND 19.30 9.20

Table S1. Conservation and dynamic changes of tumor cell phenotypes. Thec-Kit⁺Sca-1⁺ cells from spontaneous tumors were isolated by FACS sortingand serially transplanted into syngeneic B10.BR mice. Single-cellsuspensions of tumors that arose in each round were analyzed by flowcytometry using antibodies specific for CD8, Vb8, c-Kit and Sca-1. The %of cells among spleen cells are presented. N.D., not determined.

Using the medium for assaying the colony-forming units (CFU) ofhematopoeitic progenitor cells, it was possible to establish long termcultures of the TGB lymphoma cells. In over 30 passages, thec-Kit⁻Sca-1⁺ cells remained at about 0.5-1.5% of total lymphoma cellpopulation and maintained the CFU in vitro (data not shown) and tumorinitiation in vivo (Table 1, expts 3 and 4), with an undiminishedefficiency. The fact that the c-Kit+Sca-1⁺ cells remained at low %indicates that these markers must have been lost during differentiationthat occurred after the initiation of the colony formation. Thec-Kit+Sca-1− population usually disappeared during in vitro culture. Theloss of the Kit+Sca-1− cells during culture does not accompany the lossof tumor-initiation and CFU (data not shown).

Example 2 Essential Role for Up-Regulation of HIF1α Expression in theMaintenance of CSC

Having established that the c-Kit+Sca-1+ cells are CSCs in the lymphomamodel, the molecular program responsible for the self-renewal of CSCactivity was identified, using CFU as a surrogate assay. As shown inFIG. 2a , treatment with pharmacologically effective doses of Ly294002(inhibitor of PI-3 kinase-AKT signal pathway), Rapamycin (mTor-S6Kprotein synthesis pathway), SB216763 (GSK3β-beta-catenin pathway),Gö6983 (PKC inhibitor), 2-DG (hexokinase inhibitor), H89 (PKA-CREB),PDTC (NF-κB signal pathway), PD98059, SB203580, and SP600126 (MAPKfamily ERK, p38, and JNK respectively) had no effect on CFU. Incontrast, low doses of echinomycin abrogated the CFU.

In order to monitor the HIF1α activity of the CSC, a lentiviral reporterwas generated, consisting of triple HIF1 responsive elements (HRE) inthe upstream of a minimum TATA box sequence and an EGFP sequence, asshown in FIG. 2b . A pause sequence was introduced to eliminate effectof LTR promoter on the reporter. To validate the reporter, the HEK293cells were transiently transfected with either control vector or amutant HIF1α (P402A/P577A) cDNA in conjunction with either WT or mutantHRE-driven EGFP reporters. The mutation made HIF1α functional undernormoxia condition by resisting prolyl hydroxylation-mediateddegradation. As expected, the HER-EGFP reporter was specifically inducedby HIF1α but not by the control vector. In contrast, the mutant HRE EGFPreporter did not respond to HIF1α (FIG. 2b , lower left panel). Usingthis lentiviral vector, the effect of echinomycin on the % of cells withactive HIF activity was determined. As shown in FIG. 2b lower rightpanel, a distinct GFP+ population of cells that expressed both c-Kit andSca-1 markers was found (FIG. 2b and FIG. 8). The expression of GFPreflected HIF activity as it can be abrogated by mutation of the HRE(FIG. 2b , lower middle panel). The sensitivity of this subset toechinomycin was further tested. As shown in FIG. 2c , echinomycinabrogated the CSC with an IC50 of 29.4 pM, which is considerably moresensitive than other cell types, with IC50 in the nM range (Kong et al.,2005).

To substantiate the role of HIF1 activity in CSC function, the effect ofHIF inhibitors for both CFU in vitro and tumor-initiating activity invivo was tested. Since the CFU from the lymphoma CSC and normalhematopoeitic progenitor cells (HPC) can be assayed under similarconditions, the selectivity of echinomycin for HPC vs lymphoma CSC wastested. As shown in FIG. 2d , lymphoma CSC was approximately 100-foldmore sensitive to echinomycin than HPC. In serial plating experiments,the effect of echinomycin in both first and second round of CSC wastested. As shown in FIG. 9a , the CSC in both rounds were equallysusceptible to echinomycin. The specificity was confirmed by the effectof 3 different classes of HIF1α inhibitors but not by the P38 inhibitor(Fig. S9 b). It is worth noting that, at doses (5-20 pM) that had littleeffect on CFUs in the first round, the CFU in the second-round of colonyformation was significantly reduced (Fig. S9 c). These data suggest thatthe in vitro self-renewal of CSC is more addicted to HIF1α than colonyinitiation. In contrast, neither the first nor the second round ofHPC-CFU is affected by considerably higher doses of echinomycin (FIG.10).

Based on these observations, we explored the therapeutic potential ofHIF inhibitors. 1×10⁶ of cultured lymphoma cells were injected i.p. intoimmune competent B10BR mice. Four or 14 days later, the mice thatreceived lymphoma cells were either treated with vehicle only or 3 (FIG.11) or 5 (FIG. 2e ) injections of 200 ng/mouse of echinomycine at 2 dayintervals. As shown in FIG. 2e and FIG. 11, the untreated mice survivedonly 6-10 weeks, while all treated mice lived until euthanasia at 134(FIG. 2e ) or 252 days (FIG. 11) after tumor cell injection. Two otherknown HIF inhibitors, 2-methoxyestradiol (Mabjeesh et al., 2003) andGeldanamycin (Minet et al., 1999), also reduced both CFU and tumorinitiation of CSC, albeit at less efficacy (FIG. 12). The difference inefficacy may be due to different mechanisms of action andbio-availability. The therapeutic efficacy of these HIF inhibitorsdemonstrated that HIF may serve as an effective therapeutic target.

To determine the molecular mechanism for the high HIF1α activity in theCSC, the lymphoma cells were sorted into c-Kit⁺Sca-1⁺ or c-Kit⁻Sca-1⁻subsets and analyzed HIF1α, HIF-2α and HIF-3α expression by RT-PCR. Asillustrated in FIG. 3a and quantified in FIG. 3b , c-Kit⁺Sca-1⁺ cellsexpressed HIF1α at a level that is 4-fold higher than the c-Kit⁻Sca-1⁻cells. No expression of HIF2α or HIF3α was detected in the c-Kit⁺Sca-1⁺cells. Consistent with higher levels of HIF1α, expression of glucosetransporter Glut1, a known target gene of HIF1α, is also highly elevatedin the c-Kit⁺Sca-1⁺ cells. In contrast, no up-regulation of HIF1α andGlut1 was observed in the c-Kit+Sca-1+ bone marrow cells. To test if HIFactivity is selectively required for survival of the c-Kit+Sca-1+≧CSC,the tumor cell culture were treated with low doses of echinomycin (20pM) for 16 hours and analyzed the % of apoptotic c-Kit⁺Sca-1⁺ andc-Kit⁻Sca-1⁻ tumor cells. In the vehicle treated group, approximately1.8% of c-Kit⁺Sca-1⁺ and c-Kit⁻Sca-1⁻ tumor cells bound to Annexin V.Echinomycin increased Annexin V+ cells in the c-Kit⁺Sca-1⁺ tumor cellsby 6-fold, or to about 10%. No effect was observed in the c-Kit⁻Sca-1⁻tumor cells (FIG. 3c , lower panel). In three separate experimentsinvolving 10 pM of echinomycin, apoptosis of c-Kit⁺Sca-1⁺ tumor cellswas on average 3.8+0.8-fold of what was observed in vehicle control. Inthe same culture, apoptosis of the c-Kit⁻Sca-1⁻ tumor cells in theechinomycin-treated group is roughly the same (1.2+/−0.3 fold) as thevehicle group. The difference between the two groups is highlysignificant (P=0.01).

To test the general significance of HIF1α, we analyzed expression andfunction of the HIF1α in human acute myeloid leukemia (AML)-initiatingcells. Leukemia-initiating cells of AML have a phenotype of CD38⁻CD34⁺.To determine whether the HIF1α gene is over-expressed in this subset,the CD38⁻CD34⁺, CD38⁺CD34⁺, CD38⁻CD34⁻ and the CD38⁺CD34⁻ subsets weresorted by FACS (FIG. 3d ) and the expression of HIF1α and its targetGLUT1 were analyzed. As shown in FIG. 3e , the CD38⁻CD34⁺ subset had thehighest levels of HIF1α transcript. Correspondingly. GLUT1 transcriptwas also elevated in the CD38⁻CD34⁺ cells. All 6 cases of AML testedshowed increased expressions of HIF1α and GLUT1 in the CD38⁻CD34⁺ subset(data not shown), which indicate that increased HIF1α expression is ageneral feature of those bearing markers of AML-initiating cells,

The CD38⁻CD34⁺ are also known to form AML-colonies in vitro, whichprovides us with a simple assay to test the significance of HIF1α. Asshown in FIG. 6f , for all cases tested, echinomycin inhibitions colonyformation with IC50 between 50-120 pM. Although the echinomycin is alsoknown to inhibit c-Myc activity, its IC50 is in the nM range. As shownin FIG. 13, in the ranges used in this study, echinomycin stronglyinhibited HIF1α but had no effect on c-Myc function. Given the fact thatthe AML cases used have diverse genetic alterations (Table S2), thebroad inhibition by echinomycin is consistent with an important functionof HIF1α in AML-CFU, which include the CD38⁻CD34⁺ AML-initiating cells.

TABLE S2 Sample FAB type Age Rx status at Flt3 status exons Cytogeneticsenrollment 13-15 and 20 al diagnosis MI-AML-9 M4 64 untreated WT46,XY[20]^(b) MI-AML-15 M4 58 pre-treated heterozygous point mutation:46,XY[20] Y572YC in exon 14 MI-AML-32 MO 55 untreated WT 47,XY,+ 21[4];46,XY[16] MI-AML-36 M4 55 untreated WI 46,XY,t(11;19)(q23;p13.1)[20]MI-AM L-60 M-1 52 untreated Internal tandem duplication 46,XY[20] MI-AML-71 AML- 73 pre-treated WT 45,XX,−7[20] NOS

Methods

To obtain the data shown in Table S2, primers to amplify exons 5-9 ofp53, exons 13-15 and exon 20 of Flt3, exon 12 of NPM1 and exons 2-3 ofN-ras and K-ras were designed using the primer3 program (Steve Rozen andHelen J. Skaletsky (2000) Primer3 on the WWW for general users and forbiologist programmers. In: Krawetz S. Misener S (eds) BioinformaticsMethods and Protocols: Methods in Molecular Biology. Humana Press,Totowa, N.J., pp 365-386). PCR was used to amplify exons of interestusing genomic DNA from FACS-sorted blasts as template. DNA was preparedfor direct sequencing using nested sequencing primers and Exo-SAP.Mutations were identified using the Mutation Surveyor program and visualinspection of sequence tracings.

To establish the significance of HIF1α up-regulation in the c-Kit⁺Sca-1⁺cells, lentiviruses expressing HIF1α shRNA (see FIG. 14 for validationof shRNA) were first used to transduce the lymphoma cells. GFP was usedto track cells expressing the lentiviral vector. As shown in FIG. 4a ,in the vector control group with scrambled ShRNA, equal numbers ofc-Kit⁺Sca-1⁺ cells were found in GFP^(hi) and GFP^(low) subsets. Incontrast, in two shRNA-transduced tumors, the GFP^(hi) population wereessentially devoid of the c-Kit⁺Sca-1⁺ cells, which indicated that thesilencing of HIF1α abrogates the c-Kit⁺Sca-1⁺ subset. Since more than50-fold reduction of CSC was observed on day 3 after transduction, HIFactivity is required for the maintenance of the c-Kit⁺Sca-1⁺ CSC.

Consistent with this notion, after drug selection to enrich thetransduced cells, the colony formation assay revealed 70-80% reductionin the HIF1α ShRNA-transduced cells (FIG. 4b ). To test the role forHIF1α in tumor-initiating activity, control vector (with scrambledshRNA) or HIF1α shRNA-transduced tumor cells were transplanted intoB10.BR mice after three rounds of transduction. As shown in FIG. 4c ,transduction with either shRNA significantly reduced tumor-initiatingactivity as judged by the significant delay of tumor-related death.

Example 3 Down-Regulation of Vhl in CSC is Essential for Maintenance ofCSC

Since HIF1α is normally degraded under normoxia by a VHL-dependentmechanism, the expression of Vhl in the CSC was also tested. The datademonstrate an approximate 4-fold reduction in the Vhl transcripts ofc-Kit⁺Sca-1⁺ cells (FIG. 5a ). To determine the significant of Vhldown-regulation, the tumor cells were infected with Vhl-expressinglentivirus that also expresses GFP. The GFP^(hi) and GFP^(lo) subsetswere compared for the abundance of the c-Kit⁺Sca-1⁺ subset. The GFP^(hi)subset contained no c-Kit⁺Sca-1⁺ cells (FIG. 5b ). Thus, high Vhlexpression ablated the c-Kit⁺Sca-1⁺ cells. Consistent with this, theectopic expression of Vhl significantly reduced the colony formingactivity of the tumor cells (FIG. 5c ). To test the role for reduced Vhlin tumor-initiating activity, vector and VHL cDNA transduced tumor cellswere transplanted into B10.BR mice. As shown in FIG. 5d , transductionwith lentivirus expressing Vhl cDNA significantly reducedtumor-initiating activity as judged by the onset of tumor-related deathof the recipients. Taken together, the data presented in FIGS. 4 and 5demonstrate that both over-expression of HIF1α and reduction in VHL areessential for CSC activity.

Example 4 HIF Acts in Concert with the Notch Pathway in Self-Renewal ofCSC

In order to determine the underlying molecular mechanisms by which HIF1αactivation promotes self-renewal of CSC, the potential involvement ofWnt and Notch pathways was examined. Despite activation of the Wntsignaling in the TGB tumor, the data demonstrate that the dominantnegative TCF-1, which was shown to inhibit tumor growth associated withEpm2a down regulation, did not affect the CFU of the TGB CSC (data notshown). In contrast, g-secretase inhibitor, L-685, 458, an inhibitor forNotch, potently blocked the colony forming activity (FIG. 6a ). Todetermine whether the Notch signaling is over-activated in the CSC, thec-Kit⁺Sca-1⁺ cells were sorted and compared with the bulk c-Kit⁻Sca-1⁻cells for expression of the Notch target gene Hes1. As shown in FIG. 6b, sorted CSC had approximately 3.5 fold increase in expression of Hes1.In order to test whether the increased Notch activity depended on HIFactivity, three different HIF inhibitors were used to block theup-regulation of Notch targets in total TGB lymphoma cells and CSCenriched for c-Kit+ cells. The data in FIG. 6c demonstrated that all HIFinhibitors blocked expression of Hes1 among the c-Kit+ CSC.

Expression of Notch1-4 was analyzed in c-Kit⁺Sca-1⁺ and the c-Kit⁻Sca-1⁻tumor cells. As shown in FIG. 15, Notch1 and 2, but not Notch 3 and 4are expressed in the TGB tumor cells. Since two Notch genes areexpressed, a search for an effective dominant negative mutant tosuppress Notch signaling was performed. By trial and error, a potentdominant negative mutant of Notch (AA1955-2370) was identified,comprising intracellular domains of Notch1 with truncation in both N andC-termini. A nuclear localization sequence (NLS) from SV40 virus wasinserted in the N-terminus to facilitate its translocation into thenuclei. Based on the structure of Notch I-C/CSI/Mastermind/DNA complex,the deletion removed both the DNA binding RAM domain and the 4 ankyrinrepeats responsible for binding N-terminus of mastermind, whilereturning the bulk of CSL-interacting residues. As such, it is predictedto act as a dominant negative regulator of Notch signaling by preventingthe formation of mastermind-CSL-Notch IC-DNA complex. The mutant wascalled dRdA1-4dOP (FIG. 6d , left panel). As shown in FIG. 6d rightpanel, transduction of the dominant mutant resulted in about 30-foldreduction of the lies transcripts. To substantiate the role for Notchsignaling, the TGB lymphoma was transduced with either controllentiviral vector or that expressing dRdA1-4dOP. The transduced cellswere marked with GFP. As shown in FIG. 6e , in the vector control group,the % of c-Kit⁺Sca-1⁺ cells were comparable among the GFP^(hi) andGFP^(lo) cells. In contrast, dRdA1-4dOP-transduced group, more than5-fold reduction in the % c-Kit⁺Sca-1⁺ cells was observed in theGFP^(hi) subset. These data demonstrate that inactivation of the Notchpathway prevents the survival of the c-Kit⁺Sca-1⁻ cells. In serialplating experiments, transduction of dRdA1-4dOP reduced self-renewalactivity as revealed by colony formation assay (FIG. 6f ). Moreover,when the dRdA1-4dOP- or control vector-transduced cells weretransplanted into syngeneic mice, it was clear thatdRdA1-4dOP-transduction prevented the development of lymphoma, asdemonstrated by the survival analysis (FIG. 6g ).

Previous studies demonstrated that HIF1α may interact with Notchdirectly to activate its target gene, Hey2, under hypoxia conditions.Using the reporter for Hes1 promote activity, however, no significantenhancement of Notch signaling by the oxygen-resistant HIF1α mutant wasobserved (FIG. 6h, i ). Alternative explanations for the function ofHIF1α in Hes1 expression was therefore explored. It is well establishedthat, in response to Notch signaling, Hes1 expression is self-limiting,and that the negative feedback is mediated by Hes1 binding to theN-boxes in the Hes1 promoter region. Interestingly, immediately aftereach of the two critical N-boxes, a bona fide HRE was identified (FIG.6h and FIG. 16). Given their proximity, it was hypothesized that HIF1αmay directly inhibit autoregulation of Hes1. Indeed, transfection ofHes1 cDNA reduced the Hes1 promoter activity by about 10-fold. Thisinhibition was partially reversed by the oxygen-resistant HIF1α (FIG. 6j). Likewise, in the presence of Notch IC cDNA, Hes1 also repressed itsown promoter. HIF1α reversed the repression in a dose dependent manner(FIG. 6k ). Using chromatin immunoprecipitation (FIG. 6l ), significantbinding of HIF1α to the region bound by Hes1 was observed.Interestingly, the binding HIF1α and Hes1 appear to compete with eachother in binding to the region. The data suggest that HIF1α may enhanceNotch-induced Hes1 expression by antagonizing the autoregulation of theHes1.

Example 5 A Role for HIF in CSC Maintenance

The re-emergence of CSC concept relied on transplantation studies toidentify a subset of self-renewing tumor initiating cells. Since moststudies involved xenogeneic and allogeneic transplantation intoimmune-deficient host, some have suggested that the CSC concept requiresreappraisal. An important feature of the current study is to usesyngeneic immune competent mice as recipients. The self-renewingcapacity of the CSC identified in this study has been demonstrated bythree rounds of serial transplantation, in which as few as 100c-Kit⁺Sca-1⁺ cells can give rise to lymphoma in nearly 100% of therecipients. During the process, the number of c-Kit⁺Sca-1⁺ cells remainsaround 1%. While maintaining the expression of the CD8 co-receptor, thebulk lymphoma cells appear to gradually lose the expression of the Tcell receptor. In addition to giving rise to lymphoma, almost allc-Kit⁺Sca-1⁺ cells exhibit CFU activity. The data substantiate anincreasing list of genetic studies in supporting the notion of CSC,although the potential variation in tumor models with regard toexistence of CSC cannot be ruled out.

Both in vitro self-renewal and in vivo tumor initiating properties wereused to characterize the molecular mechanism of self-renewal of CSC. Inboth assays, the role for HIF1α was demonstrated by drug inhibition,shRNA silencing and over-expression of oxygen-dependent HIF inhibitorVHL. Since the expression of transduced vectors leads to almostimmediate disappearance of the CSC population, and since the short-termtreatment (12 hours) of echinomycin resulted in a specific reduction ofthe c-Kit⁺Sca-1⁺ cells by increased apoptosis, the HIF1α is likelynecessary for survival of CSC.

The increased HIF activity in the murine lymphoma is caused by bothover-expression of HIF1α and down-regulation of Vhl. Since Vhl isresponsible for the oxygen-mediated degradation of HIFa, the increasedHIFa activity no longer requires hypoxic environment. In addition, itshould be noted that in the 6 cases AML samples tested, we have notobserved increased expression of VHL (data not shown). Yet the HIF areactive based on expression of its target and sensitivity to echinomycin.Therefore, additional mechanisms likely exist to allow oxygen-resistantfunction of HIF in AML-CFU. The effect of low doses of echinomycin onall AML samples tested suggest that the mechanism described herein maybe generally applicable for tumors grown in area with high levels ofblood supplies, including leukemia and lymphoma. For areas of solidtumors with poor blood supplies, the mechanism can be operative evenwithout HIF over-expression or VHL down-regulation.

Example 6 A Molecular Pathway for Maintenance of CSC

In investigating the molecular pathway responsible for the maintenanceof CSC, enhanced activity of Notch pathway was observed, as revealed byincreased expression of Notch target genes, in the CSC in comparison tothe bulk tumor cells. The data demonstrate that all three HIF inhibitorstested block Notch activation in the c-Kit⁺ subset. Interestingly, theinhibition appear specific for the c-Kit⁺ subset of TGB lymphoma, whichis enriched for CSC, as the drugs had no effect on the Notch targetexpression if the total TGB lymphoma cells were used. The significanceof Notch in CSC maintenance and tumor development is demonstrated byeffective ablation of the CSC and tumorigenesis by an ectopic expressionof a dominant inhibitor of Notch signaling, dRdA1-4dOP in the TGB tumorcells. Again, the selective ablation demonstrates that Notch signalingis specifically required for the maintenance of CSC, while the survivalof the bulk tumor cells is independent of Notch signaling. Takentogether, these data demonstrate that HIF maintains CSC by regulatingNotch signaling.

Hes1 is an important Notch target known to be critical forstem/progenitor cell functions. The data described herein indicate thatHIF1α potentiated the induction of Hes1 by Notch. In contrast with theprevious studies using Hey2 promoter as readout, no direct co-operationbetween HIF1α and Notch IC in the induction of the Hes1 gene wasobserved. Rather, it was shown that HIF1α prevents the negative-feedbackauto-regulation of the Hes1 gene by inhibiting its binding to theN-boxes in the Hes1 promoter. Given the general, although notnecessarily universal, role of Notch in maintenance of a variety oftissue stem cells, the data indicate an important functionalconservation between CSC and tissue stem cells.

Example 7 The HIF Pathway Plays a Role in CSC Function

An important way to validate the role for HIF pathway in the CSCfunction was to test whether HIF inhibitor echinomycin can be used totreat AML in a xenogeneic model. Studies using two AML samples have beenperformed. 6×10⁶ AML cells (either AML71-PB, which had poor prognosisbased on cytogenetics data, or AML-15-PB which had moderate prognosisbased on cytogenetics; see Table S2 above) from blood were transplantedinto sublethally irradiated NOD-SCID mice. Starting at 2 weeks aftertransfer, half of the recipient mice received three injections of 200ng/mouse/injection of echinomycin, once every other day. After two weeksof pause, these mice received 3 more injections. The other half of themice were left untreated as control. At 7 weeks after transplantation,all untreated mice in both groups (3 mice per group) became moribund,while all echinomycin treated mice were healthy. Analysis of theperipheral blood and bone marrow of AML-71PB recipients is shownprovided in FIG. 17.

As shown in FIG. 17A, the untreated group had an average of 12% humanCD45⁺ cells in the blood. Echinomycin treatment completely wiped outhuman CD45⁺ cells. A clearly definable human cell population wasobserved in the bone marrow of untreated bone marrow, but not with theechinomycin treatment (FIGS. 17B and C). Further analysis of the humanCD45⁺ cells indicated that they were immature, as judged by their lackof CD14 and CD15 marker (FIG. 17C lower right panel). Moreover, a highproportion of the human CD45⁺ cells exhibited marker of AML stem cells(CD34⁺CD38⁻), as indicated in FIG. 17C (upper right panel). These datademonstrated the feasibility of the xenogeneic model and criticalsupport for treating hematologic cancer such as AML using echinomycin.These data help demonstrate that HIF1α activity is required for themaintenance of stem cells for hematological malignancies and that HIFserves as a valuable therapeutic target for cancer therapy. Moreimportantly, the data establish that the same principle applies to humanAML.

Example 8 Therapeutic Elimination of Leukemia Stem Cells for AML

This example demonstrates the therapeutic potential of HIF inhibitorsfor human hematological malignancies. AML was used as a model becausethe phenotype of AML leukemia stem cells (AML-LSC) waswell-characterized, and because AML-LSC function in vivo can be assayedusing an established xenogeneic model. AML-LSC have the phenotype ofCD34⁺CD38⁻. To determine whether the HIF1α gene is over-expressed inthis subset of cells, CD34⁺CD38⁻, CD34⁺CD38⁺, CD34⁻CD38⁻ and CD34⁻CD38⁺subsets were sorted by FACS (FIG. 18a ), and the expression of HIF1α andits target GLUT1 were analyzed. As shown in FIG. 18b , the CD34⁺CD38⁻subset had the highest levels of HIF1α transcript. Correspondingly,GLUT1 transcript was also elevated in the CD38⁻CD34⁻ cells. All 6 casesof AML tested showed increased expressions of HIF1α and GLUT1 in theCD38⁻CD34⁻ subset (data not shown), which indicate that increased HIF1αexpression is a general feature of those cells bearing markers ofAML-LSC cells.

CD34⁻CD38⁻ cells are also known to form AML-colonies in vitro, therebyproviding a simple assay to test the significance of HIF1α. As shown inFIG. 18c , for all cases tested, echinomycin inhibited colony formationwith an IC50 between 50-120 pM. Although the echinomycin is also knownto inhibit c-Myc activity, its IC50 for c-Myc is in the nM range. Thebroad inhibition by echinomycin is consistent with an important functionof HIF1α in AML-CFU, which include the CD34⁺CD38⁻ AML-LSC.

Conventional cancer therapy appears to enrich cancer stem cells. An NFκBinhibitor, dimethylamino analog of parthenolide, showed some selectivityfor AML-LSC. Since the HIF1α activity appears to be selectivelyactivated in AML, AML-LSC might be selectively targeted by echinomycin.As shown in FIG. 18d , low doses of echinomycin selectively inducedapoptosis of the CD34⁺CD38⁻ population, in comparison to the dominantAML cells (CD34⁺CD38⁺ for most but CD34⁻CD38⁺ for two AML cases).

A xenogeneic AML model using human AML samples was established to testwhether echinomycin can be used as a potential therapeutic agent forAML. Primary clinical samples from AML patients reconstituted irradiatedNOD.SCID mice with immature human myeloid cells were characterized bythe expression of human CD45, CD11b, but not mature myeloid markers CD14and CD15. Remarkably, a short term treatment with echinomycin startingat 15 days after transplantation completely eliminated human cells fromsample AML 150 and dramatically reduced the burden of human leukemia ofAML 71 (FIG. 18e ). The residual cells in echinomycin treated mice werenot differentiated as judged by the lack of mature myeloid markers CD14,and CD15 (FIG. 18f ).

The incomplete remission of AML71 enabled a determination of whetherechinomycin selectively reduces the AML-LSC, using the CD34⁺CD38⁻markers. Echinomycin reduced the percentage of CD34⁺CD38⁻ cells in bonemarrow by more than 10-fold (FIG. 18g , top panel). Even among the humanCD45⁺ compartment, the relative abundance of AML-LSC was also reduced(FIG. 18g , lower panel). To substantiate the impact of echinomycin onthe leukemia initiating cells, serial transplantation studies werecarried to determine whether echinomycin reduced self-renewal ofAML-LSC. As shown in FIG. 18h , while bone marrow from untreated miceinduced leukemia in all new recipients, bone marrow from echinomycintreated mice failed to do so in any recipients. Therefore, the residualcells in the echinomycin-treated bone marrow were devoid of AML-LSC.

Example 9 Experimental Procedures

Materials and Methods for Examples 1-7 are disclosed below.

AML Samples

AML patients diagnosed at the University of Michigan ComprehensiveCancer Center between 2005 and 2009 were enrolled into this study. Thestudy was approved by the University of Michigan Institutional ReviewBoard. Written informed consent was obtained from all patients prior toenrollment. The same AML diagnostic criteria (>=20% myeloblasts in thebone marrow or peripheral blood) were used and determined FABsubclassification through review of both laboratory and pathologyreports dated at the time of diagnosis and interpreted byhematopathologists. Cytogenetic risk stratification was determinedaccording to SWOG/ECOG criteria.

Antibodies, Flow Cytometry, and Immunofluorescence

Fluorochrome-conjugated antibodies specific for CD1117 (c-kit) andLy-6A/E (Sca-1) were purchased from either e-Bioscience (Ja Jolla,Calif.), while those specific for CD8 and Vβ8 were purchased from BectonDickinson-Pharmingen (Ja Jalla, Calif.). The cell surface markers wereanalyzed by flow cytometry using LSRII (Becton Dickinson, Mountain View,Calif.). The specific subsets were sorted by FACSAria.

RT-PCR and Real-Time PCR

Expression of HIF1α, HIF-2α, HIF-3α, VHL, and Glut1 was determined byRT-PCR and real-time PCR. The primers used were HIF1α, forward,5′-agtctagagatgcagcaagatctc-3′ (SEQ ID NO: 1); reverse,5′-tcatatcgaggctgtgtcgactga-3′ (PCR)(SEQ ID NO: 2),5′-ttcctcatggtcacatggatgagt-3′ (real-time PCR)(SEQ ID NO: 3); hif-2a,forward, 5′-cgacaatgacagctgacaaggag-3′ (SEQ ID NO: 4); reverse,5′-ttggtgaccgtgcacttcatcctc-3′ (SEQ ID NO: 5); hif-3a, forward,5′-atggactgggaccaagacaggtc-3′ (SEQ ID NO: 6); reverse,5′-agcttcttctttgacaggttcggc-3′ (SEQ ID NO: 7); vhl, forward,5′-tctcaggtcatcttctgcaac-3′ (SEQ ID NO: 8); reverse,5′-aggctccgcacaacctgaag-3′ (SEQ ID NO: 9); Glut1, forward5′-tgtgctgtgctcatgaccatc-3′ (SEQ ID NO: 10) and reverse5′-acgaggagcaccgtgaagat-3′ (SEQ ID NO: 11); mHes1F: gccagtgtcaacacgacaccgg (SEQ ID NO: 12), mHes1R: tcacctcgttcatgcactcg (SEQ ID NO:13); HIF1αF, 5′-ccatgtgaccatgaggaaatgagag-3′ (SEQ ID NO: 14); HIF1αR,5′-tcatatccaggctgtgtcgactgag-3′ (SEQ ID NO. 15); GLUT1F,5′-tcaatgctgatgatgaacctgct-3′ (SEQ ID NO: 16); GLUT1R,5′-ggtgacacttcacccacataca-3′ (SEQ ID NO: 17).

ShRNA-Mediated Knockdown of HIF1α and Ectopic Expression of VHL anddRdA1, 2dOP

Lentiviral vectors carrying siRNAs are known in the art. The coresequence of HIF1α-ShRNA-1 is 5′-ctagagatgcagcaagatc-3′ (SEQ ID NO: 18),while that of HIF1α-ShRNA-2 is 5′-gagagaaatgcttacacac-3′ (SEQ ID NO:19). The same vector was used to express full length VHL cDNA, anddominant negative Notch mutant dRdA1-42dOP (AA1995-2370). The tumor cellcultures were infected with either control lentivirus or lentivirusencoding HIF1α shRNA, dRdA1-42dOP or VHL cDNA by spinoculation. Thecultures were selected with 5 μg/ml of blasticidin for 5 days to removeuninfected cells.

In vitro colony formation assay for tumor cells and bone marrow cells isknown in the art.

In Vivo Tumorigenicity Assay

Given numbers of total tumor cells or sorted subsets were injected intoeither immune competent B10.BR mice or RAG-2^(−/−)BALB/c mice. Moribundmice were considered to have reached experimental endpoint and wereeuthanized. The therapeutic effects were analyzed by Kaplan Meiersurvival analysis.

The invention claimed is:
 1. A method of treating acute myeloidleukemia, comprising administering echinomycin as a sole active agentand a pharmaceutically acceptable carrier to a human in need thereof, ata dose and interval to maintain a serum concentration of echinomycinthat kills cancer stem cells without adversely affecting normalhematopoiesis, wherein the maintained serum concentration of echinomycinis less than or equal to 5 nM.
 2. A method of treating acute myeloidleukemia, consisting of administering a composition consisting ofechinomycin and a pharmaceutically acceptable carrier to a human in needthereof, wherein the echinomycin is administered at a dose and intervalto maintain a serum concentration of echinomycin of less than or equalto 5 nM.
 3. The method of claim 2, wherein the maintained serumconcentration is 0.01 nM to 5 nM.
 4. The method of claim 3, wherein themaintained serum concentration is 0.1 nM to 5 nM.
 5. The method of claim1, wherein the maintained serum concentration is 0.01 nM to 5 nM.
 6. Themethod of claim 5, wherein the maintained serum concentration is 0.1 nMto 5 nM.